1. Field of the Invention
The present invention relates to three novel genes of SEQ ID Nos. 1-3 useful for water-stress tolerance in biological systems, wherein said genes are differentially expressed in Tea plant under drought conditions and a method of introducing said genes into a biological system to help develop water stress tolerance.
2. Prior Art
Crop performance is sensitive to a number of biotic and abiotic factors, wherein drought stress constitutes an important yield-limiting determinant. Drought stress in context to the present invention refers to the situation when the amount of water in the plant is not sufficient to meet the transpirational requirements of the plant that leads to altered visible symptoms such as leaf curling. Drought should also be quantifiable through an important physiological parameter, leaf water potential (a measure of water status within the leaf tissue). Plant response to water deficit is dependent on the amount of water lost, the rate of loss, the duration of drought stress, the plant variety/species under consideration, developmental stage of the plant, and other environmental variables such as temperature, relative humidity etc.
Stress affects many metabolic pathways and structures, which may be the result of some up or down-regulated genes. Many of the water deficit induced genes encode gene products predicted to protect cellular function. One often noticed response of the plant is the accumulation of metabolically compatible solutes such as proline, glycine betaine, pinitol, carnitine, mannitol, sorbitol, polyols, trehalose, sucrose, oligosachharides and fructans in large quantities. These are chemically dissimilar and are excluded from the surface of the proteins, thus keeping the proteins preferentially hydrated. Accumulation of these compounds results in decreased water potential thus, facilitating water movement in the cell and helps in maintaining the turgor, a mechanism proposed to safeguard against water deficit.
These compounds have capability to (a) stabilize the membranes and other macromolecules such as nucleic acids and proteins, and can function as scavenger of free radicals. Indeed the transgenic plants over-expressing the genes responsible for the synthesis of these compounds were found to be more tolerant as compared to the wild types under the situation of water deficiency. Classical studies include: (a) transgenic tobacco overexpressing SacB gene (encoding levan-sucrase) from Bacillus subtilis accumulated fructan several folds that showed significantly greater growth and dry weight accumulation in response to drought stress (Pilon-Smits, E. A. H., Ebskarnp, M. J. M., Paul, M. J., Jeuken, M. J. W., Weisbeek, P. J. and Smeekens, S. C. M. (1995) Improved performance of transgenic fructan-accumulating tobacco under drought stress. Plant Physiol. 107:125-130); (b) Transgenic tobacco overexpressing P5CS (Δ1-pyrroline-5-carboxylate synthetase; the enzyme involved in the proline biosynthesis from L-glutamate via Δ1-pyrroline-5-carboxylate) from mothbean (Vigna aconitifolia) lead to 10-18 fold increase in proline content and showed better growth under water stress compared to the wild type (Kavi, K. P. B., Hong, Z., Miao, G-H., Hu C. A. A. and Verma, D. P. S.(1995) Plant Physiol. 108:1387-1394); (c) Transgeric tobacco expressing TPS1 gene (synthesizing trehalose-6-phosphate synthase) from yeast accumulated trehalose and showed better drought tolerance compared to the wild types (Holmstorm, K. O., Mantyla, E., Mandal, W. A., Palva, E. T., Tunnela, O. E. and Londesborough J. (1996) Drought tolerence in tobacco. Nature. 379: 683-684); (d) Transgenic tobacco overexpressing betB gene (synthesizing betaine aldehyde dehydrogenase) from E. coli showed better performance under osmotic stress conditions (Holmstrom, K. O., Welin, B. and Mandal, A. (1994) Production of the Escherichia coli betaine-aldehyde dehydrogenase an enzyme required for the synthesis of the osmoprotectant glycine betaine, in transgenic plants. Plant J. 6:749-758); (e) Transgenic tobacco expressing imtl gene (synthesizing myo-inositol-o-methyl transferase and involved in D-ononitol biosynthesis) from Mesembryanthemum crystallinum showed more adaptation to water stress (Sheveleva, E., Chmara, W., Bohnert, H. J. and Jensen, R. G. (1997) Increased salt and drought tolerence by D-Ononitol production in transgenic Nicotiana tabacum. Plant Physiol.5: 1211-1219); (f) Production of some of these osmo-protectants under drought stress is mediated through the plant hormone abscisic acid (ABA).
Recently, 9-cis-epoxycarotenoid dioxygenase gene (NCED), involved in ABA synthesis has been found to be strongly induced under water deficit in the 8-day-old cowpea plants (Iuchi, S., Kobayashi., Yamaguchi-Shinozaki, K. and Shinozaki, K. (2000) A stress-inducible gene for 9-cis-epoxycarotenoid dioygenase involved in abscisic acid biosynthesis under water stress in drought-tolerant cowpea. Plant physiol. 123:553-562). NCED mRNA was found to be increased both In reply to: water stressed leaves and roots of tomato (Thompson, A. J., Jackson, A. C., Parker, R. A., Morpeth, D. R., Burbidge, A. and Taylor, I. B. (2000) Abscisic acid biosynthesis in tomato: regulation of dioxygenase mRNA by light/dark cycles, water stress and abscisic acid. Plant. Mol. Biol. 42:833-845).
Apart from the osmolytes assisting in maintaining the hydration status, drought or osmotically stressed plants, synthesize several genes, which produce water channel proteins and water transport proteins such as membrane proteins of family aquaporins that can alter the cellular water potential and thus, protect against water deficit (Chrispeels, M. J. and Agre, P. (1994) Water channel proteins of plants and animal cells. Trends in Biochem Sci. 19:421-425; Bohnert H, J. and Jensen, R. G. (1996).
Strategies for engineering water-stress tolerance in plants. TIBTECH. 14:89-97; Johansson, I., Larsson, C., Ek B. and Kjellbom, P. (1996) The major integral proteins of spinach leaf plasma membranes are putative aquaporins and are phosphorylated in response to Ca and apoplastic water potential. The Plant Cell. 8:1181-1191. Accumulation of LEAs to high concentrations also coincides with the acquisition of desiccation tolerance. One of the groups-3 of LEA proteins is predicted to play a role in the sequestration of ions that are concentrated during cellular dehydration. Another group-5 of LEA proteins are predicted to sequester ions during water loss. The maintenance of total water potential during water deficit can be achieved by osmotic adjustment. Two proteins, osmotin and nonspecific lipid transfer proteins, are stress induced and are thought to play a role in controlling pathogens. Nonspecific lipid transfer proteins are induced by drought (Plant A. L., Cohen, A., Moses, M. S. and Bray, E. A. (1991) Nucleotide sequence and spatial expression pattern of a drought abscisic acid induced gene in tomato. Plant Physiol. 97:900-906; Toress-Schumann, S., Godoy, J. A. and Pintor-Toro, J. A. (1992) A probable lipid transfer protein is induced by NaCl in stems of tomato plants. Plant Mol. Biol.18:749-757).
Heat shock proteins that are induced by water deficit (Borkird, C., Simoens, C., Villarroel, R. and VanMontagu M. (1991) Gene associated with water-stress adaptation of rice cells and identification of two genes as hsp 70 and ubiquitin. Physiol. Plant. 82: 449-457; Almoguera, C. and Jordano, J. (1992) Developmental and environmental concurrent expression in sunflower dry-seed-stored low-molecular-weight heat shock protein and Lea mRNAs. Plant Mol. Biol. 19:781-792) may be involved in refolding of proteins in order to regain their function, or the prevention of protein aggregation (Vierling E. (1991) the roles of heat shock proteins in plants. Annual review of Plant Physiol. and Plant Mol. Biol. 42:579-620) during drought.
Small HSPs are another type of proteins those have been associated with plant desiccation tolerance. Small HSPs might act as molecular chaperones during seed dehydration and first few days of rehydration (Hoekstra, F, A., Golovina, E, A. and Buitink, J. (2001) Mechanisms of plant desiccation tolerance. Trends in Plant Science. 6(9): 43-439). OsHSP110 accumulated in shoots of rice seedlings in response to salinity, drought and low temperature apart from heat shock. It has been shown that two of the hsps, hsp70 in maize and hsp27 in soybean can also be induced by water stress (Sachs, M. M. and David Ho, T. H. (1986). Alterations of gene expression during environmental stress in plants. Ann. Rev. Plant Physiol. 37: 363-376). Most of the changes in gene expression occur during dehydration and thus many dehydration-specific gene products have been isolated but very few rehydration-specific proteins are known (Bernacchia, G., Schwall, G., Lottspeich, F., Salamini, F., and Bartels, D. (1995) Molecular Characterization of the Rehydration process in the Resurrection Plant Craterostigma Plantagineum. EMBO J 14: 610-618).
Complex regulatory and signaling processes, most of which are not understood, control the expression of genes during water deficit. Genes involved in two types of protein degrading mechanisms, proteases and ubiquitin are induced by water deficit. The gene products may be involved in degradation of proteins that are denatured during cellular water loss. Also, thiol protease an enzyme involved in degradation of proteins that have been denatured by stress, is induced by water deficit (Guerrero, F. D., Jones, J. T. and Mullet, J. E. (1990) Turgor-responsive gene transcription and RNA levels increases rapidly when pea shoots are wilted: sequence and expression of three inducible genes. Plant Mol. Biol. 15: 11-26).
Neale, A. D., Blomstedt, C. K., Bronson, P., Le, T.-N., Guthridge, K., Evans, J., Gaff, D. F. and Hamill, J. D. (2000. The isolation of genes from the resurrection grass Sporobulus stapfianus which are induced during severe drought stress. Plant, Cell and Environment. 23:265-277) isolated drought stress induced genes from resurrection grass Sporobolus stapfianus. Detected genes were found to encode an eIF1 translation initiation factor, two drought stress-inducible glycine-rich proteins, a tonoplast-intrinsic protein (TIP) and an early light-inducible protein (ELIP). Previously, no such gene products have been found to be associated with drought stress. This is the first report suggesting that a gene encoding an eLF1 translation initiation factor may have a role in the drought stress response of plants.
Several different stresses may trigger the same or similar signal transduction pathways. The plant hormone ABA also accumulates in response to the physical phenomenon of loss of water caused by the different stresses, and elevation in endogenous ABA content is known to induce certain water-deficit induced genes. Therefore, ABA accumulation is a step in one of the signal transduction pathways that induces genes during water deficit. Various protein kinases have been reported in plants and are thought to function in phosphorylation processes in various signal transduction pathways, including water-stress and ABA responses.
A cDNA, pKABA1, corresponding to a protein kinase, which is induced by ABA, has been isolated (Anderberg, R. J. and Walker-Simmons, M. K. (1992) Isolation of wheat cDNA clone for an abscisic acid-inducible trascript with homology to protein kinases.
Proc. Natl. Acad. Sci. USA 89: 10183-10187). A new homoebox-containing gene, Athb-12 and Athb-7 are induced by water deficit and exogenous ABA treatment but time course experiment have shown that both of these are regulated in a different manner (Lee, Y. H. and Chun. J. Y. (1998) A new homeodomain-leucine zipper gene from Arabidopsis thaliania induced by water stress and abscisic acid treatment. Plant Mol. Biol. 37: 377-384).
Available evidences suggest that stress induced responses may be ABA mediated or independent of ABA (Shinozaki, K. and Yamaguchi-Shinozaki, K. (1997) Gene expression and signal transduction in water-stress response. Plant Physiol. 115: 327-334). ABA mediated gene response may require or may not require protein synthesis to take place. The induction of mRNA of rd22 gene by ABA, which showed homology to an unidentified seed protein of Vicia faba, required protein synthesis to take place since cycloheximide inhibited induction of the gene (Yamaguchi-Shinozaki K. and Shinozaki, K. (1993) The plant hormone abscisic acid mediates the drought-induced expression but not the seed-specific expression of rd22, a gene responsive to dehydration stress in Arabidopsis thaliana. Mol. Gen. Genet. 238:17-25).
Structure analysis of the gene revealed the presence of regulatory sequences (cis-acting motif) asl (TGACG; SEQ ID NO: 4) and sp1 (GGGCGG; SEQ ID NO: 5) at −463 and −443 positions, respectively (Briggs, M. R., Kadonaga, J. T., Bell, S. P. and Tijan, R. (1986). Purification and Biochemical characterization of the promoter-specific transcription factor, Spl. Science 234:47-52; Lam, E., Benfey, P. N., Gilmartin, P. M., Fang, R. X. and Chua N-H. (1989). Site specific mutations alter in vitro factor binding and change promoter expression pattern in transgenic plants. Proc. Natl. Acad. Sci. USA. 86: 7890-7894). Also, were present the sequences that resembled myb (a family of transcription factors with Tip cluster motif) recognition elements TGGTTAG (SEQ ID NO: 6) at −144 and −666 and 2 bHLH (basic helix-loop-helix; MYC) recognition elements (CACATG; SEQ ID NO: 7) at −200 and −191 position. A cDNA (rd22BP1) encoding a MYC related DNA binding protein was isolated, which was found to encode a 68 kD protein that has a typical DNA binding domain of a basic region helix-loop-helix leucine zipper motif in MYC-related transcription factors.
The protein indeed binds to the MYC recognition site (Abe, H., Yamaguchi-Shinozaki, K., Urao, T., Iwasaki, T., Hosokawa, D. and Shinozaki, K. (1997) Role of Arabidopsis MYC and MYB Homologs in Drought- and Abscisic Acid-Regulated Gene Expression. The Plant Cell. 9:1859-1868). A drought and ABA inducible gene has also been cloned that encodes MYB-related protein ATMYB2. Both rd22BP 1 (MYC) and ATMYB2 (MYB) proteins were shown to function as transcription activators in the dehydration and ABA-inducible expression of the rd22 gene (Abe, H., Yamaguchi -Shinozaki, K., Urao, T., Iwasaki, T., Hosokawa, D and Shinozaki, K. (1997). Role of Arabidopsis WC and MYB Homologs in Drought- and Abscisic Acid-Regulated Gene Expression. The Plant Cell. 9:1859-1868).
In contrast to rd22 in Arabidopsis, HVA22 gene in barley is induced in response to drought and ABA, but is also induced in the presence of cycloheximide. The promoter region of HVA22 contains ABA responsive complex ABRE3, CE1 and another ABA responsive complex that relies on the interaction of a G-box with another yet unidentified coupling element (Shen, Q. and Ho, T-H D. (1995) Functional Dissection of an Abscisic Acid (ABA)-Inducible gene Reveals Two Independent ABA-Responsive Complexes Each Containing a G-Box and a novel cis-Acting Element. The Plant Cell. 7:295-307)
Yamaguchi-Shinozaki K and Shinozaki, K. (1993. Characterization of the expression of dessication-responsive rd29 gene of Arabidopsis thaliana and analysis of its promoter in transgenic plants. Mol. Gen. Genet. 236: 331-340) cloned a dehydration responsive gene rd29A that was independent of ABA responsive pathway. The sequence TACCGACAT (SEQ ID NO: 8) was found to be regulating the genes induced under drought conditions and was found in the promoter regions of other dehydration inducible genes.
Upon over-expression of DREBIA (a dehydration responsive element binding protein) under the control of rd29a promoter in A. thaliana, a number of stress tolerant genes were expressed and resulted in an improved tolerance under drought and several other stresses (Kasuga, M., Liu, Q., Miura, S., Yamaguchi-Shinozaki, K. and Shinozaki, K. (1999) Improving plant drought, salt, and freezing tolerance by gene transfer of a single stress-inducible transcription factor. Nature Biotechnology. 17:287-291).
Analysis of another gene of DRE-binding protein DREB2 showed that its promoter was induced under water stress in transgenic arabidopsis (Nakasimha, K., Shinwari, Z. K., Sakuma, Y., Seki, M., Miura, S., Shinozaki, K. and Yamaguchi-Shinozaki, K.(2000) Organization and expression of two Alabidopsis DREB2 genes encoding DRE-binding proteins involved in dehydration and high salinity responsive gene expression. Plant. Mol. Biol. 42:657-665). These genes do not require ABA for their expression, but do respond to exogenous ABA.
There are also drought inducible genes that do not respond to ABA treatment. These include rd 21, erd1, and rd 19 that code for thiol proteases, CIp protease and thiol protease, respectively (Shinozaki, K. and Yamaguchi-Shinozaki, K. (1997) Gene expression and signal transduction in water-stress response. Plant Physiol. 115:327-334). Indeed, the information on such genes is very scarce.
There is always a need and search for novel drought related genes so that better adaptation may be sought. Apart from the genes and gene sequences listed in the Table 1, the novel gene sequences may be listed as follows:
ABRE. ABA-responsive element (PyACGTGGC; SEQ ID NO: 9) (Shen Q and Ho (1995) Functional Dissection of an Abscisic Acid (ABA)-Inducible gene Reveals Two Independent ABA-Responsive Complexes Each Containing a G-Box and a novel cis-Acting Element. The Plant Cell. 7:295-307).
G-box, ubiquitous regulatory elements (CACGTG; SEQ ID NO: 10). (Menkens, A. E., Schindler, U. and Cashmore A. R. (1995) The G-box: ubiquitous regulatory DNA element in plants bound by GBF family of bZIP proteins. Trends in Biochem Sci. 20:506-510).
DRE, Dehydration-responsive element (TACCGACAT; SEQ ID NO: 11) (Shinozaki, K. and Yamaguchi-Shinozaki, K. (1996) Molecular responses to drought and cold stress. Current opinion in biotechnology. 7:161-167). MYBRS, MYB recognition sequence (PyAACPyPu; SEQ ID NO: 12) (Urao T, Yamaguchi-Shinozaki K, Urao S, Shinozaki K (1993) An Arabidopsis myb homolog is induced by dehydration stress and its gene product binds to the conserved MYB recognition sequence. The Plant Cell 5:1529-1539).
MYCRS, MYC recognition sequence (CANNTG; SEQ ID NO: 13) (Abe, H., Yamaguchi-Shinozali, K., Urao, T., Iwasaki, T., Hosokawa, D and Shinozaki, K. (1997) Role of Arabidopsis MYC and MYB Homologs in Drought- and Abscisic Acid-Regulated Gene Expression. The Plant Cell. 9: 1859-1868).
While working with gene(s) and gene fragments (gene fragment in context to the present invention refers to partial nucleotide sequences of the complete gene), related to drought or other stresses, the following are possibilities:
(a) Gene can be Cloned Through Several Routs as Shown Below in Table 1.
TABLE 1Route/TechniqueUsedReferenceProtein sequencingWeretilnyk, E. A. and Hanson, A. D. 1990. Molecular cloning offollowed by oligo-a plant betenin oldohydo dehydrogenase, an enzyme implicatednucleotide synthesisin soaptation to salinity and drought Prod. Notl. Acad. Sci. IISAand screening87: 2745-2749.Plaque hybridizationNakashima, k. Shinwari, Z. K., Sakuma, Y., Seki, M., Miura, S.,Shipozaki, K and Yamaguchi-Shinozaki, K. 2000. Organizationand expression of two Arapldopsts DRED2 genes eneedingDRE binding proteins involved in dehydration and high salinityresponsive gene expression Plant. Mol. Biol. 42. 657-665.PCR based cloningHirayama, T., Ohto, C., Mizoguchi, T., and Shinozaki, K. 1995.A gene encoding a phosphoinositol-specific phospholipase C ininduced by dehydration and salt stress in Arabidopsis thalianaProc. Natl Acad. Sci. 92: 3903-3907Library screening usingRichard, S., Morency, M., Drevet, C., Jouanin, L., and Seguin, S.heterologous probe2000. Isolation and characterization of a dehydrin gene fromwhite spruce induced upon wounding, drought and cold stress.Plant Mol. Biol. 43: 1-10.Gene cloning usingRoberts, J. K. and Key, J. L. 1991. Isolation and characterizationheterologous probeof a soybean hsp 70 gene. Plant molecular biology, 16: 671-683.Differential ScreeningChang, S., Puryear, J. D., Dias, A. A. D. L, Funkhouser, E. A.,Newton, R. J., and Calway, J. 1996. Gene expression underwater dificit in lobiolly pine (Pinus taeda): isolation andcharacterization of cDNA clones. Physiol. Plant. 97: 139-148.MicroarraySeki, M., Nerusaka, M., Abe, H., Kasuga, M., Yamaguchi-Shinozaki, K., Caminci, P. Hayashizaki, Y., and Shinozaki, K.2001 Plant Cell 113: 61-72Subtractivea. Lee, S. W., Tomasetto, C., and Sagar R, 1991. Positivehybridizationselection of fumous suppression genes by subtractivehybridization Proc. Nati Acad. Sci. USA, 88: 2825-2829.b. Buchanan-Wollaston, V. and Ainaworth, C, 1997 Leafsenescence in Brassica naus cloning of senescence relatedgene bu substractive hybridication Plant Mol. Biol. 33, 821-834.(b) Gene Cloned from Organisms can be Expressed in Other Organisms.
As has been shown by Kishor, Kavi. P. B. R. Hong, Z., Miao, G. H., Hu, C. A. and Verma, D. P. S. (1995 Overexpression fo pyrroline-5-carboxylate synthetase increases proline production and confers osmotalerence in transgenic plants. Plant Physiol. 108:1387-1394 and the references therein) that the gene pyrroline-5-carboxylase synthetase was cloned from Vigna aconotifolia and expressed into tobacco through transgenic technology Transgenic tobacco plants were more tolerant under water stress conditions.
Pilon Smits, E. A. H., Ebskamp. M. J. M., Paul, M. J. Jeuken, M. J. W., Weisbeek, P. J. and Smeekens. Improved performance of transgenic fructan-accumulating tobacco under drought stress. Plant. Physiol. 107:125-130) transferred SacB gene from Bacillus subtilis into tobacco and found increased drought tolerance.
Holmstrom, K. O., Welin, B. and Mandal, A. (1994, Production of the Escherichia coli betaine-aidehyde sakydrogonase an enzyme required for the synthesis of the osmoprotectant glycine betaine, in transgenic plants. Plant J. 6:749-758) transferred betaine-aldehyde dehydrogenase from Escherichia coli (a microorganism) into tobacco (higher plant) and found to be drought tolerant.
(c) Genes Expressed in Response to Drought Stress can be Expressed by Other Environmental Variables as Well.
Iuchi, S., Kobayashi, Yamaguchi-Shimuzaki, K and Shinozaki, Kazuo (2000 A stress-inducibe gene for 9-cis-epoxycarotenoid dioxygenase involved in abscisic acid biosynthesis under water stress in drought tolerant compound. Plant physical 123:553-662) reported the expression of VcNCED1 in response to water and salt stress.
Pelloux J., Jolivet, Y., Hontaine, V., Banvoy, J., and Dizengromel, P. (2001 Changen in Rubieco and Rubisco activase gene expression and polypeptide expression.
Richard. S. Mordancy. M. Drevet. C. Jouanin, L. and Seguin, S. (2000. Isolation and characterization of a dehydrin gene from white spruce induced upon wounding, drought and cold stress. Plant Mol. Biol. 43: -1-10} reported a gene PgDhnl. which was induced In repose to drought, cold stress and upon wounding.
Nakashima. K. Shinwari, Z. K. Sakuma, Y. Seki. M. Miura, S, Shinozaki, K. and Yamaguchi-binozaki K. (2000 Organization and expression of two Arabidopsis DREB2 genes encoding DRE-binding proteins involved in dehydration and high salinity responsive gene expression. Plant. Mol, Biol 42; 657-665) reported the expression of drought responsive element DREB2 genes in response to dehydration and high salinity stress.
Hirayama. T. Ohto. C. Mizoguchi. T. and Shinozaki, K. (1995. A gene encoding a phosphoinositol-specific phospholipase C in induced by dehydration and salt stress in Arabidopsis thaliano, Proc. Natl. Acad. Sci 92: 3903-3907) reported expression of phosphoinosltol-specific phospholipase C in response to drought, salinity and low temperature.
Weretilnyk. E. A., and Hanson. A. D. (1990. Molecular cloning of a plant betaine-aldehyde dehydrogenase, an enzyme implicated in adaptation to salinity and drought. Proc. Natl. Acad. Sci., USA, 87: 2745-2749) reported the expression of betaine-aldehyde dehydrogenase gene in response to drought as well salinity,
D. Identified Gene may be Used to Study Regulatory Elements. Regulatory Elements in Context to Present Invention Relate to the Regions such as Promoters, Transcriptional Factors and Other Sequences which Control the Expression of the Gene.
Using stress regulated gene HVA1. Straub, P. P. Shen Q. and Ho, Tuan-hua. D. (1994. Structure and promoter analysis of an ABA- and stress-regulated barley gene, HVA1. Plant. Mol. Biol. 26: 617-630) analysed promoter of the gene, Michel, D., Salamini F., Bartels, D. Dale, P., Baga, M. and szalay, A. (1993. Analysis of a desiccation and ABA-responsive promoter Isolated from the resurrection plant Craterostigma plantagineum Plant Journal 4: 29-40) selected drought responsive gene CdeT27-46 and analysed its promoter region.
Urao T, Yamaguchi-Shinozaki K. Urao S. Shinozaki K. (1993. An Arabidopsis myb homolog is induced by dehydration stress and its gene product binds to the conserved MYB recognition sequence. Plant Cell 5: 1529-1539) identified the sequences encoding transcription factors in a dehydration responsive gene Atmyb2 Yamaguchi-Shinozaki. K. and Shinozaki, K. (1997, Characterization of the expression of a desiccation-responsive rd29 gene of arabidopsis thaliana and analysis of its promoter in transgenic plants. Mol Gen Genet 236: 331-340) analysed the promoter region of a drought inducible gene rc(29.
Abe. H., Yamaguchi-Shinozaki. K. Urao, T. Iwasaki, T. Hosokawa. D and Shinozaki. K. (1997. Role of Arabidopsis MYC and MYB Homologs in Drought- and Abscisic Acid-Regulated Gene Expression. The Plant Cell.9: 1859-1868) analysed a drought inducible gene rd22 for regulatory factors.
Shen Q and Ho T. D. (1995. Functional Dissection of an Abscisic Acid (ABA)-Inducible gene Reveals Two Independent ABA-Responsive Complexes Each Containing a G-Box and a novel c/s-Acting Element. The Plant Cell. 7: 295-307) analysed HVA22 gene for regulatory elements and reported novel coupling elements.
e. Gene or Gene Fragment Isolated from One System can be Used as a Probe to Study the Similar Genes in Other Plant Systems
Roberts, J K and Key. J. L. (1991. Isolation and characterization of a soybean hsp70 gene. Plant molecular biology, 16: 671-683) used hsp70 gene cloned from Drosophila to clone the similar gene from soybean.
Singia. S. L. Pareek A and Grover. (1997 Yeast HSP104 homologue rice HSP 110 is developmental- and stress regulated Plant science, 125: 211-219) showed that yeast hsp KM and rice hsp 110 are very similar. These are expressed in response to desiccation, salinity, low temperature and high temperature.
Shen, Q. Chen, C. N. Brands. A. Pan. S. M. and Ho, T. D. (2001 The stress- and abscisic acid-induced barley gene HVA 22: developmental regulation and homologues in diverse organisms. Plant Molecular Biology. 45: 327-340) reported a drought inducible gene HVA 22 in several organisms such as cereals, arabidopsis. Caenorhabitis elegans. man. mouse, and yeast.
(f) Gene Expressed in One Organ can be Expressed in Organ as Well as Shown Below in Table 2:
TABLE 2OrganReferenceRoots andNemoto, Y., Kawakami, N., and Sasakuma. 1999. Isolationleavesof novel early salt-responding genes from wheat (Triticumaestivum L.) by differential display. Theor. Appl. Genet 98:673-678Thompson, A. J., Jackson, A. C., Parker, R. A., Morpeth, D. R.,Burbidge, A. and Taylor, I. B. (2000) Abscisic acidbiosynthesis in tomato: regulation of dioxygenase mRNAby light/dark cycles, water stress and abscisic acid.Plant.Mol.Biol. 42: 833-845Sheaths andClaes, B., Dekkkeyser, R., Villarroel, R., Bulcke, M. V. D., Bauw, G.,rootsMontagu, M. V., and Caplan, A. 1990. Characterization of a ricegene showing organ specific expression in response to salt stressand drought. Plant Cell, 2: 19-27.Stem TissueRichard, S., Morency, M., Drevet, C., Jouanin, L., and Seguin, S.and partially2000. Isolation and characterization of a dehydrin gene from whiteexpandedspruce induced upon wounding, drought and cold stress, Plant Mol.vegetative buds,Biol.43: 1-10reproductivebuds(g) It is possible to Clone Full Length cDNAs or Genomic DNA by Using Standard Protocols as detailed by Ausubel. F. M. Brend R. Kingston. R. E., Moore. D. D. Seidman. J. G. Smith, J. A., Struhl. K. 1987. Current protocols in molecular biology. Publisher John Wiley and Sons. New York: and Sambrook. J. Fritsch, E. F, and Maniatis. T. 1989. Molecular cloning; a laboratory manual, Second edition. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
A summary of various drought-related genes is given below in Table 3.
TABLE 3List of drought related genes along with their source and the predicted function.PredictedSpecies from whichGENESrole/HomologyisolatedREFRENCEA1494Cysteine thiolArabidopsis thalianaWilliams et al.,protease1994 Plant Mol.Biol. 25: 259-270ADHAlcohol″de Bruxelles et al.,dehydrogenase1996 Plant Physiol.111: 381-391;Dolferus et al.,1994 Plant Physiol.105: 1075-1087;Jarillo et al., 1993Plant Physiol. 833-837Athb-7Homeodomain″Söderman et al.,leucine zipper1996 Plant Journaltransfactor10: 375-381Athb-12Homeodomain″Lee and Chun, 1998leucine zipperPlant Mol. Biol. 37:transfactor377-384AthH2Aquaporin″Kaldenhoff et al.,1993 Plant Mol.Biol. 23: 1187-1198AthK1Histidine kinase″Urao et al., 1998FEBS Left. 427:175-178CDPK1/K2Cal dependent″Urao et al., 1994protein kinaseMol. Gen. Genet.244: 331-340HSP70-1/ERD2HSP-cognate″Kiyosue et al., 1994Plant Mol. Biol. 25:791-798HSP81-2/ERD8HSP-cognate″Kiyosue et al., 1994Plant Mol. Biol. 25:791-798rd22Unidentified seed″Yamaguchi-protein of ViciaShinozaki andfabaShinozaki., 1993Mol. Gen. Genet.238: 17-25RAB18Dehydrin″Lang et al., 1994Plant Physiol. 104:1341-1349; Langand Palva, 1992Plant Mol. Biol. 20:951-962RD19Cysteine protease″Koizumi et al.,1993 Gene 129:175-182RD28, RD21Cysteine protease″Yamaguchi-Shinozaki et al.,1992 Plant CellPhysiol. 33: 217-224rd29A, rd29BDrought responsive″Iwasaki et al., 1997promoter element,Plant Physiol. 115:drought related1287; Wang et al.,genes1995 Plant Mol.Biol. 28: 605-617DREB1ADehydration″Kasuga et al, 1999responsiveNatureelements bindingBiotechnology. 17:proteins287-291Tps1Trehalose″Holmstrom et al.,biosynthesis1994 Plant Journal6: 749-758RPK1Receptor-like″Hong et al., 1997protein kinasePlant Physiol 113:1203-1212cAtP5CSΔ1-pyrroline-5-″Yoshiba et al., 1995carboxylatePlant J. 7: 751-760synthetaserd19A; rd21ACysteine proteases″Koizumi et al.,1993 Gene129: 175-182UBQIUbiquitin extension″Kiyosue et al., 1994proteinPlant Mol. Biol. 25:791-798cATCDPK1; cATCDPK2CA2+-dependent,″Urao et al., 1994calmodulin-The Plant Cell 5:independent1429-439protein kinasescAtPLC1Phosphatidylinositol-″Hirayama et al.,specific1995 Proc. Natl.phospholipase CAcad. Sci. USA 92:3903-3907ERD11; ERD13Glutathione S-″Kiyosue et al., 1993.transferasesBiochem. Biophys.Res. Comm.196: 1214-1220cAtsEHSoluble epoxide″Kiyosue et al., 1994hydrolasePlant J. 6: 259-269kin2Similarity to″Kurkela & Borg-animal antifreezeFranck 1992 PlantproteinsMol. Biol. 29: 689-692pA1494Similarity to″Williams et al.,proteases1994 Plant Mol.Biol. 25: 259-270ERD1Similar to a Clp″Kiyosue et al., 1993ATP-dependentBiochem. Biophys.protease subunitRes. Comm.196: 1214-1220Athsp70-1Similar to the″Kiyosue et al.,HSP70 heat-shock-1994 Plant Mol.protein familyBiol. 25: 791-98Athsp81-2similar to the″Kiyosue et al., 1994HSP81 heat-shockPlant Mol. Biol.protein family25: 791-98rd22Similar to an″Iwasaki et al., 1995unidentified seedMol. Gen. Genet.protein from Vicia247: 391-398fabalti65, lti78Unknown″Nordin et al., 1993Plant Mol. Biol. 21:641-653pRABAT1D11 LEA-protein″Lang & Palva,related1992 Plant Mol.Biol. 20: 951-962Atmyb2MYB-protein-″Urao et al., 1993relatedThe Plant Cell 5:transcription factor1429-1439ERD10; ERD14D11 LEA-protein″Kiyosue et al., 1994relatedThe Plant CellPhysiol. 35: 225-231SacBFructosylBacillus subtilisPilon-Smits et al.,transferase1995 Plant Physiol.107: 125-130MC12LKR/SDH cDNABrassica napusDeleu et al., 1999of A. thlianaPlant Cell andEnvironment 22:979-988MC43His-3 linker″Deleu et al., 1999protein/ribosomalPlant Cell andprotein S12Environment 22:979-988pBN115Similar toBrassica napusWeretilnyk et al.,polypeptides1993 Plant Physiol.encoded by pBN19101: 171-177and pNB26 (B. napus),and COR15(A. thaliana)BnD22Similar to proteaseBrassica napusDowning et al.,inhibitors1992 Plant J. 2:685-693VuNCED1ABA biosynthesisCowpeaIuchi et al., 2000Plant Physiol. 123:553-562GapC-CratCytosolicCraterostigma plantagineumVelasco et al., 1994glyceraldehyde 3-Plant Mol. Biol. 26:phosphate541-546dehydrogenasepSPS1Sucrose-phosphate″Ingrams & Bartels,synthase1996 Annu RevPlant Physiol 47:377-403PSS1; pSS2Sucrose synthases″Ingrams & Bartels,1996 Annu RevPlant Physiol 47:377-403pcC 37-31Similar to early-″Bartels et al., 1992light-inducibleEMBO J. 11: 2771-2778proteinspcC 13-62Unknown″Piatkowski et al.,1990 Plant Physiol.94: 1682-1688pcC 27-04D11 LEA-protein″Piatkowski et al.,related1990 Plant Physiol.94: 1682-1688pcC 6-19D11 LEA-protein″Piatkowski et al.,related1990 Plant Physiol.94: 1682-1688pcC 3-06D7 LEA-protein″Piatkowski et al.,related1990 Plant Physiol.94: 1682-1688pcC 17-45D95 LEA-protein″Piatkowski et al.,related1990 Plant Physiol.94: 1682-1688pcECP40D11 LEA-proteinDaucus carotaKiyosue et al., 1993relatedPlant Mol. Biol.21: 1053-1068Bet BGlycine betaineEscherichia coliHolmstrom el al.,biosynthesis1994 Plant Journal6: 749-758pTS.6Plasma membraneGlycine maxSurowy and Boyer,H+-ATPase1991 Plant. Mol.Biol 16: 251-262SC514Lipoxygenase″Bell and Mullet1991 Mol. Gen.Genet. 230: 456-462Ha hsp17.6HaLow-molecular-Helianthus annuusCoca et al., 1994hsp17.9weight heat-shockPlant Mol. Biol. 25:proteins479-492Ha ds 10D19 LEA-protein″Almoguera andrelatedJordano1992 PlantMol. Biol. 19: 781-792Ha ds11D113 LEA-protein″Almoguera andrelatedJordano, 1992 PlantMol. Biol. 19: 781-792B8; B9; B17D11 LEA-proteinHordeum vulgareClose et al., 1989relatedPlant Mol. Biol. 13:95-108B19.1; B19.3; B19.4D19 LEA-protein″Espelund et al., 1992relatedThe Plant CellEnviron. 18: 943-949HVA22LEA″Shen et al., 2001(Lateembryogenesis-Plant Mol. Biol. 45:abundant) and RAB327-340(responsive to ABA)BLT4Similar to protease″Dunn et al., 1991inhibitorsMol. Gen. Genet.229-389-394pBADBetaine aldehydeHordeum vulgareIshitani et al., 1995dehydrogenaseMol. Gen. Genet.247: 391-398pcht28Acidic endochitinaseLycopersicon chilenseChen et al., 1994Mol. Gen. Genet.145: 195-202SAM1; SAM3S-adenosyl-L-Lycopersicon esculentumEspartero et al., 1994methioninePlant Mol. Biol. 25:synthetases217-227P31Cytosolic copper/zinc″Perl-Treves andsuperoxide dismutaseGalun 1991 PlantMol. Biol. 17: 745-760TSW12A lipid transfer″Torres-Schumann etproteinal., 1992 Plant Mol.Biol. 18: 749-757pLE16Similar to lipid″Plant et al., 1991transfer proteinsPlant Physiol. 97:900-906pLE4D11 LEA-protein″Cohen et al., 1991relatedPlant Physiol. 97:1367-1374pUM90-1Similar to MsaciAMedicago sativaLuo et al., 1992 J.and pSM2075Biol. Chem. 267(22):polypeptides15367-15374pSM1075Similar to MsaciA″Luo et al., 1991 Plantand pUM90-1Mol. Biol. 17: 1267-1269polypeptidesMsaciASimilar to pUM90-1″Laberge et al., 1993and pSM2075Plant Physiol.polypeptides101: 1411-1412pPPC1PhosphoenolpyruvateMesembryanthemumVernon et al., 1993carboxylasecrystallinumThe Plant CellEnviron. 16: 437-444pRAB 16AD11 LEA-proteinOryza sativaMundy & Chua 1988.relatedEMBO J. 7: 2279-2286salTUnknown″Claes et al., 1990 ThePlant Cell 2: 19-27Apx1 geneCytosolic ascorbatePisum sativumMittler and Zilinskasperoxidase1994 Plant J. 5: 397-405Sod 2 geneCytosolic copper/zinc″White and Zilinskassuperoxide dismutase1991 Plant Physiol.96: 1291-129226gSome similarity to″Guerrero et al., 1990aldehydePlant Mol. Biol.dehydrogenase15: 11-267aSimilar to channel″Guerrero et al., 1990proteinsPlant Mol. Biol. 15:11-2615aSimilarity to″Guerrero et al., 1990proteasesPlant Mol. Biol. 15:11-26pLP2S-AdenosylPinus taedaChang et al., 1996methioninePhysiol. Plant. 97:synthatase139-148pLP3Silk fibrion and rat″Chang et al., 1996chondroitin corePhysiol. Plant. 97:protein139-148pLP4Tomato protein TMA″Chang et al., 1996SN1(water deficitPhysiol. Plant. 97:inducible)139-148pLP5Copper binding″Chang et al., 1996proteinPhysiol. Plant. 97:139-148P22Similar to proteaseRaphanus sativusLopez et al., 1994inhibitorsPhysiol. Plant. 91:605-614H26D11 LEA-proteinStellaria longipesRobertson andrelatedChandler 1992 PlantMol. Biol. 19: 1031-1044pMA2005D71 LEA-proteinTriticum aestivumCurry et al., 1991relatedPlant Mol. Biol.16: 1073-1076pMA1949D7 LEA-protein″Curry & Walker-relatedSimmons 1993 PlantMol. Biol. 21: 907-912EmD19 LEA-protein″Litts et al., 1987relatedNucleic Acids Res.15: 3607-3618PKABAIProtein kinase″Aderberg andWalker-Simmons1992 Proc. Natl.Acad. Sci. USA89: 10183-10187Pmbm1L-isoaspartyl″Mudgett & Clarkemethyltransferase1994J.Biol.Chem.269:25605-25612M3 (RAB-17)D11 LEA-proteinZea maysClose et al., 1989relatedPlant Mol. Biol. 13:95-108pMAH9Similar to RNA-″Gómez et al., 1988binding proteinsNature 334: 262-264
Below is specifically given a state of art knowledge with reference to cloning of drought stress related genes.
Reference may be made to document (1) by Yamaguchi-Shinozaki, K. and Shinozaki, K. (1994) The Plant Cell. 6: 251-264, wherein is described the identification of a novel cis-acting element involved in responsiveness to drought, low temperature; or high salt stress from a model plant Arabidopsis. 
Reference may be made to document (2) by Li, L. g., Li, S. f., Tao, Y., and Kitagawa, Y. (2000) Plant Science 154: 43-51, wherein a novel water channel protein was cloned from rice which was shown to be involved with the chilling tolerance in Xenopus oocytes. 
Reference may be made to document (3) by Tabaeizadeh; Zohrer,; Yu; Long-Xi; Chen; Ri-Dong, U.S. Pat. No. 5,656,474 dated Aug. 12, (1997) wherein two osmotic stress- and ABA-responsive members of the endochitinase gene family were isolated and identified from the leaves of drought-stressed Lycopersicon chilense plants.
Reference may be made to document (4) by Kim; Soo Young U.S. Pat. No. 6,245,905 dated Jun. 21, (2001) wherein a nucleic acid molecule encoding the Abscisic acid responsive element binding factor 2 (ABF2) was isolated that binds abscisic acid responsive elements in plants.
Reference may be made to document (5) by Kim; Soo Young U.S. Pat. No. 6,218,527 dated Apr. 17, (2001) wherein a nucleic acid molecule encoding the Abscisic acid responsive element binding factor 3 (ABF3) was isolated that binds abscisic acid responsive elements in plants.
Reference may be made to document (6) by Thomshow; Michael F.; Stockinger; Eric, J. U.S. Pat. No. 5,892,009 dated Apr. 6, 1999 wherein a gene designated as CBF1, encoding a protein CBF1, which binds to a region regulating expression of gene which promote cold temperature and dehydration tolerance in plants was cloned.
Reference may be made to document (7) by Chun; Jong-Yoon; Lee; Yong-Hun. U.S. Pat. No. 5,981,729 dated Nov. 9, 1999 wherein a novel gene induced by water deficit and abscisic acid was cloned.
The Drawbacks in the Prior Art are:
    a. Earlier work to clone the genes related to drought stress focused on model plant system and mainly annuals. Perennial evergreen plants such tea experience several rounds of drought stress during their life cycle. The plant is, therefore, expected to harbor novel gene(s) imparting tolerance to drought.    b. There is always search of novel genes so as to exploit it for generating more drought tolerant plants. Model plants such as Arabidopsis thaliana and other domesticated plants as mentioned in Table 1 have been used to clone the drought-related genes. Novel genes can be expected from a hitherto unstudied plant.    c. Methods reported to clone drought related gene relied on differential screening of cDNA library, analysis of differential cDNA library, and subtractive hybridization (Tables 1, 2 and 3). These have inherent limitation of using two samples at a time for analysis Therefore, after identification and cloning of differentially expressed genes, these used to be tested for their expression analysis during recovery and/or in response to other variables such as salt stress/ABA treatment etc. Therefore, appropriate technology needs to applied in order to focus on the desired gene at the beginning itself.
The above drawbacks have been eliminated for the first time in a simple and reliable manner by the present invention, which is not so obvious to the person skilled in the art.